NVS-ZP7-4 inhibits hepatocellular carcinoma tumorigenesis and promotes apoptosis via PI3K/AKT signaling

NVS-ZP7-4 was identified as a novel chemical reagent targeting the zinc input protein ZIP7, which accounts for the zinc surge from the apparatus to the cytoplasm. Since zinc dysregulation is related to multiple diseases, in this study, we aimed to identify the anti-tumor effects of NVS-ZP7-4 and explore the molecular mechanisms of NVS-ZP7-4 in hepatocellular carcinoma (HCC) progression. We found that NVS-ZP7-4 inhibited cell viability, caused cell cycle arrest, induced apoptosis, and inhibited the proliferation, migration, and invasion of HCCLM3 and Huh7 cells. We further investigated the inhibited activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was involved in the antitumor effect of NVS-ZP7-4 in HCC. Furthermore, NVS-ZP7-4 inhibited HCC tumor growth in vivo. The present study demonstrated that NVS-ZP7-4 is a promising therapeutic target for HCC by regulating PI3K/AKT signaling.

www.nature.com/scientificreports/ To the best of our knowledge, the treatment efficiency and underlying mechanisms of NVS-ZP7-4 have not been explored in the field of HCC; therefore, we aimed to reveal the anti-tumor functions of NVS-ZP7-4 and its underlying mechanisms in HCC treatment for the first time and provide a novel promising treatment for HCC.

Methods
All methods were performed in accordance with relevant guidelines and regulations.
Cell culture. Huh7 (adult hepatocellular carcinoma), HCCLM3 (adult hepatocellular carcinoma) cells were used for this study and purchased from the Chinese Academy of Sciences (Shanghai, China). The cells were incubated with Dulbecco's modified Eagle's medium (DMEM; Gibco, USA), 10% fetal bovine serum (FBS; Gibco, USA), and 10% penicillin-streptomycin solution at 37 °C, 5% CO 2 and standard humidity. Mouse primary hepatocytes were isolated using BALB/c nude mouse liver according to a reported protocol 32 . Firstly, liver was perfused using Ca 2+ -and Mg 2+ -free Hanks' balanced salt solution (HBSS, Sigma) through the portal vein. Secondly, liver was perfused with 0.1% collagenase I solution (Sigma) in HBSS containing Ca 2+ and Mg 2+ . A few minutes letter, liver was excised and dispersed into cold HBSS. The cell suspension was generated and filtered through 70-μm pore size nylon cell strainer (Sigma). The cell supernatant was centrifuged and pellets were resuspended in DMEM with 10% FBS. Cells were cultured for further experiments.
Cell viability assay and cell proliferation. To determine the cell viability after NVS-ZP7-4 treatment, cell counting kit-8 (CCK-8) was used to measure the viability of Huh7, HCCLM3 cells and mouse primary hepatocytes after different treatments. Cells were seeded in 96-well plates (5 × 10 3 /well) and treated with NVS-ZP7-4 or DMSO for 24 h. CCK-8 solution was then added to each well (10 μL/100 μL) and incubated for 2 h at 37 °C. The absorbance was measured at a wavelength of 450 nm. Cell viability For cell proliferation, cells were seeded 96-well plates (5 × 10 3 /well) and treated with different reagents. After 0 h, 24 h, 48 h, 72 h, cells were added with CCK-8 solution (10 μL/100 μL) and incubated for 2 h at 37 °C. The absorbance was measured at a wavelength of 450 nm.
Apoptosis assays. The cells were treated with NVS-ZP7-4 or DMSO for 24 h before apoptosis detection.
Cell suspensions were collected and stained with annexin V and propidium iodide (PI) in the dark for 15 min, and the cells were then analyzed with a flow cytometer.
Cell cycle staining assay. Cells were seeded in 6-well plates and treated with various concentrations of NVS-ZP7-4 or DMSO for 24 h. Cell suspensions were collected, stained with PI, and analyzed using cell flow cytometry.
Colony formation assay. Cells were seeded at 1,000 cells/well into six-well plates for the colony formation assay. After approximately 2 weeks of culture, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Visible colonies (diameter > 0.1 mm) were photographed and counted. EdU assay. A 5-ethynyl-2′-deoxyuridine (EdU) assay (RIB-BIO) was conducted according to the manufacturer's instructions. Briefly, cells were inoculated in a 96-well plate, incubated with 50 nM EdU-A reagent for 2 h, and then fixed and dyed using Apollo solution and Hoechst3342 solution. A fluorescence microscope AXIO Vert A1 (Carl Zeiss AG) was used for the photography.
Migration and invasion assays. Only for transwell invasion assays, the upper chambers were coated with Matrigel film (Corning, USA) at 37 °C overnight before use. Both for transwell migration and invasion assays, cells were pretreated with NVS-ZP7-4 or DMSO for 24 h and resuspended in DMEM. Suspensions (200 μL) were re-seeded into the upper chamber, and 750 μL of DMEM with 10% FBS was added to each well. The cells were fixed with 4% paraformaldehyde after 48 h. Cells crossing the membrane were recorded using an EVOS XL Core Instrument (AMEX1,000, Thermo Fisher Scientific, USA) at 100 × and 200 × magnifications.
Western blot analysis. Cells were inoculated in 6-well plates and treated with different reagents for 24 h.
Then, the cells were collected and lysed for 30 min with RIPA buffer (Beyotime, Shanghai, China). The supernatant was harvested after centrifugation at 1.5 × 10 4 rpm for 20 min. The protein concentration was detected using a BCA kit (Beyotime) and boiled with loading buffer (Beyotime) for 5 min. SDS-PAGE gels (12% or 10%) were used for protein electrophoresis, and the proteins were transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked and incubated with antibodies, and Super Signal West Atto (Thermo Scientific, MA, USA) were used for signal detection. China) were raised under standard conditions of temperature, light, and free access to water and food. Mice were subcutaneously injected with 100 μL of Huh7 cells (2 × 10 6 cells) to establish a subcutaneous HCC mouse model. The mice were randomly divided into two groups (n = 5): the control group received intraperitoneal injections of DMSO (0.1% in PBS, 2 mL/kg) three times a week, and the NVS-ZP7-4 group received NVS-ZP7-4 (1 mg/kg) three times a week. The mice were anesthetized with pentobarbital and sacrificed 2 weeks later. Tumor and body weights were recorded, and tumor tissues were collected for further immunohistochemical analysis. The study was approved by the Institutional Research Ethics Committees of Affiliated Tumor Hospital of Xinjiang Medical University (K-2021024). Mice were sacrificed by cervical dislocation. The study was reported in accordance with ARRIVE guidelines. Statistical analysis. For in vitro and in vivo examinations, data are presented as the mean ± standard deviation, with at least three repeats. ANOVA was used to measure the differences among groups, with a significant p value of < 0.05. All analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA).

Ethical approval. The study was approved by the Institutional Research Ethics Committees of Affiliated
Tumor Hospital of Xinjiang Medical University (K-2021024).  Fig. 1. Figure 1C and D shows that HCCLM3 and Huh7 cells displayed decreased cell viability after NVS-ZP7-4 treatment in a dose-dependent manner. Interestingly, cytotoxicity analysis showed that NVS-ZP7-4 was relatively less cytotoxic to mouse primary hepatocytes, suggesting the hypo-toxicity of NVS-ZP7-4 to normal liver cells (Fig. 1E). For further experiments, we used concentrations of 0.5 and 1 μM in HCCLM3 and Huh7 cells, and used 0.08% DMSO as control. The effect of NVS-ZP7-4 on the proliferation of HCCLM3 and Huh7 cells was detected using colony formation and EdU assays. The colony number and number of EdU-positive cells were significantly inhibited in a dose-dependent manner (Fig. 2), suggesting that NVS-ZP7-4 significantly inhibited HCC cell growth.

NVS-ZP7-4 induced HCC cell apoptosis.
The PI3K/AKT pathway can prevent the programmed death of tumor cells and inhibit apoptosis, thus promoting the survival of tumor cells. Firstly, the apoptosis-positive cells were significantly induced in a dose-dependent manner as shown in Fig. 3A and B. Secondly, p-ZIP7 was significantly repressed after NVS-ZP7-4 treatment in HCCLM3 and Huh7 cells, but failed to regulate the expression of ZIP7 in cells. And then we examined the effect of NVS-ZP7-4 on cleavage of PARP1, which is part of the apoptotic cascade. Anti-apoptotic proteins within cells, such as BCL2, must be overwhelmed, and BAX should be activated to induce apoptosis. Consistently, as shown in Fig. 3C, D HCCLM3 and Huh7 cells treated with NVS-ZP7-4 had significantly decreased levels of PARP1, caspase-3, and BCL2 protein, as well as enhanced levels of cleaved caspase-3, and BAX protein expression. These data indicate that NVS-ZP7-4 activates caspasedependent apoptosis in HCCLM3 and Huh7 cells. Besides, NVS-ZP7-4 treatment in vivo induced the expression of cleaved caspase-3 in a xenograft nude mouse model (Fig. 7D).

NVS-ZP7-4 inhibited HCC in vivo.
BALB/c nude mice were established to mimic the therapeutic efficacy of NVS-ZP7-4 in HCC. The in vivo results showed that NVS-ZP7-4 treatment significantly inhibited Huh7 tumor weight (Fig. 7A-C). The expression of PCNA is strongly related to DNA synthesis as a cofactor for DNA polymerase δ, thus remarkably correlating with the status of cellular proliferation. In our study, decreased PCNA expression in xenograft tumor tissues was also detected in the xenograft model treated with NVS-ZP7-4, suggesting that NVS-ZP7-4 significantly inhibited proliferation in vivo (Fig. 7D). These results implied that NVS-ZP7-4 is a potential therapeutic agent for the treatment of HCC.

Discussion
HCC is the fastest growing cause of cancer-related death in the USA, with the highest mortality and incidence in East Asia and Africa; thus, it is crucial to develop a valid treatment strategy for HCC 10,11 . NVS-ZP7-4 was first discovered to inhibit ZIP7 in T cell acute lymphoblastic leukemia and was further investigated as a novel www.nature.com/scientificreports/ treatment for malignant tumors 3 . Our data demonstrated that NVS-ZP7-4 inhibits HCC growth both in vitro and in vivo, and may be a promising therapeutic target for HCC. Continuous proliferation, permanent replication, and activated invasion and metastasis are essential hallmarks of cancer, which equip cells with malignancy 33 . HCC also shares the vital characteristics of rapid proliferation and a high degree of cancer-related metastasis 10 . In our study, we revealed that NVS-ZP7-4 inhibited cellular proliferation, invasion, and migration of Huh7 and HCCLM3 cells, which supports the therapeutic efficiency of NVS-ZP7-4. We also showed that NVS-ZP7-4 causes G1 phase arrest and induces apoptosis, which might contribute to its growth inhibition. Cancer resistance to apoptosis is essential in tumorigenesis; thus, targeting the apoptosis process might provide several novel therapies in oncology 33 . For example, the combination of X-linked inhibitor of apoptosis antisense oligonucleotide (AEG35156) with the first-line targeted drug sorafenib resulted in a moderate increase in progression-free survival and overall survival compared with sorafenib treatment alone 34 . However, although the mechanisms of both intrinsic and extrinsic pathways of apoptosis have been elaborated recently, it is still difficult to identify and select core apoptotic proteins for efficient targeted therapies 35,36 ; thus, novel treatments must be investigated for further preclinical and clinical screening. In our study, we confirmed the induction of apoptosis after NVS-ZP7-4 treatment, which was consistent with a previous study in human colorectal cancer cells with knockdown of the targeted protein ZIP7 19,20 . The enhanced apoptosis due to NVS-ZP7-4 might be related to the elevated ER stress, since studies in multiple diseases also identified triggered ER stress after ZIP7 removal via genetic knockdown [37][38][39] , which finally overloaded the ER, triggering apoptosis 38 .
The PI3K/AKT signaling network participates in numerous feedback loops, counteracts various signaling, and provides ample opportunities to circumvent the effects of chemotherapy. Thus, combining drugs targeting the PI3K/AKT pathway is important for addressing chemotherapeutic resistance and establishing novel tumor treatments 40 . Significantly, activation of PI3K/AKT signaling was proven to be a major event in hepatocarcinogenesis [41][42][43] . In our study, we confirmed that NVS-ZP7-4 treatment functioned through inhibition of the PI3K/AKT pathway. Considering the important role of zinc as a second messenger, the inhibition of PI3K/ AKT signaling might be attributed to the decreased zinc in the cytoplasm after NVS-ZP7-4 usage 31 . Similarly, overexpression of the NVS-ZP7-4 targeted protein ZIP7 caused activation of the AKT signaling pathway in gastric and breast cancer 31,44,45 .
Small molecules targeting the PI3K/AKT pathway, including PI3K/mTOR, pan-PI3K, and isoform-selective PI3K inhibitors, have been undergoing a large number of preclinical and clinical trials to investigate their efficacy and safety 40 . For example, a PI3K inhibitor suppressed cell proliferation and induced apoptosis in anlotinibresistant osteosarcoma (OS) models and supports the PI3K inhibitor clinically used in anlotinib-refractory   48 . However, with most HCC patients diagnosed in the advanced stage of the disease, treatment options are limited and lead to poor prognosis. Chemotherapy is an important tool, but it also has a high rate of drug resistance 49 . The only approved first-line therapy with sorafenib has shown limited survival benefits and poor tolerability 10 . Notably, our in vivo experiments identified that NVS-ZP7-4 treatment of HCC in nude mice decreased tumor weight, further implying its therapeutic potential by repressing the PI3K/AKT pathway in HCC. To the best of our knowledge, this is the first study to confirm the therapeutic role of NVS-ZP7-4 in HCC    www.nature.com/scientificreports/ www.nature.com/scientificreports/ and identify that NVS-ZP7-4 acts as an inhibitor targeting PI3K/AKT. Therefore, our study provides potential prospects for independent or combined treatment of HCC with NVS-ZP7-4. However, we noticed that there are some limitations in our study. Firstly, we have not proved the direct mechanisms underlying NVS-ZP7-4, so more experiments are needed in the future to verify its effectiveness. Secondly, we should explore the heterogeneity of HCC treatment of NVS-ZP7-4 based on different cell lines and provide more information for clinical application.

Conclusion
In conclusion, our study verified the effects of NVS-ZP7-4 on HCC cell proliferation, migration, invasion, apoptosis, and cell cycle progression. Furthermore, NVS-ZP7-4 inhibited the activation of PI3K/AKT signaling, and in vivo, NVS-ZP7-4 treatment of HCC in nude mice resulted in decreased tumor weight and expression of